A variety of techniques and devices are commercially available for the detection and measurement of substances present in fluid or other translucent samples by determining the light transmitivity of the sample. In particular, a number of photometric devices are capable of simultaneously performing individual assays on a plurality of liquid or other translucent samples. Such methods generally handle a multiplicity of samples by using "microplates" which contain a standard array (8.times.12) of wells and which are made of a optically transparent material. The optical density of the samples is measured by determining attenuation undergone by light as it passes through the translucent samples, contained in the microplate wells, to conventional photodetecting means.
A widespread use for microplates is in the enzyme-linked immunosorbent assay (ELISA) technique which is used for detection and quantitation of an extensive range of substances and biological cells in academic research and biotechnology as well as for clinical testing. In such assays, molecules of a marker enzyme (such as alkaline phosphatase) are deposited on the bottom and part of the way up the sides of each of the wells of a microplate; each well having been assigned to interact previously, directly or indirectly, with a sample containing an analyte of interest. The number of marker enzyme molecules bound to each well of the plate is a function of the concentration of analyte in the sample of interest. Determination of the activity of the bound enzyme, therefore, permits detection or quantitation of the analyte.
For determination of fluid-phase enzyme activity, current techniques for both research and clinical applications employ kinetic analysis which involves measurement of the initial rate of enzymecatalyzed, chromogenic reactions in the presence of excess of the enzyme substrate; a procedure which has several well-known advantages over the alternative "end-point" analysis method of allowing the enzyme to react with a chromogenic substrate for a fixed period of time and then making a single optical density measurement after quenching the enzymes. In kinetic analysis, multiple readings are made within the initial (typically linear) reaction period and the intervals between readings are necessarily short (typically less than 30 seconds). By using kinetic analysis, the introduction of errors caused by (a) differences in initial optical density and/or (b) loss of independence from substrate concentration, is substantially avoided.
Currently available automated optical density measurement instruments for microplates typically function by mechanically moving either the multi-well microplate or the optical components themselves in order to successively perform assays of samples located at the plurality of individual assay sites. This requirement places a severe restraint on the time required to actually measure the transmittance in all wells of the microplate, thereby making large scale kinetic analysis assay applications impractical due to the extended sampling times. In part because of this, "end-point" analysis is employed for ELISA protocols read by current instruments.
A measurement system which is capable of reading a plurality of assay sites in sequence without relative physical movement of the microplate and the optical components is disclosed in Wertz et al. U.S. Pat. No. 4,408,534 which discloses the use of fiber optic transmission means with a single light source sequentially coupled to a plurality of optical fibers which transmit light to the measurement sites. However, the apparatus described in the Wertz patent uses a highly inefficient system for coupling light from the light source into the optical fibers, which in turn leads to a variety of potential problems for kinetic measurements of enzyme activity. For example, the Wertz apparatus requires a high power light source and the increased light intensity can adversely affect the chemical reactions in the assay sites by increasing the operating temperature of the measurement system non-homogeneously and hence altering the rates of reaction in different wells to a different extent. In addition, such systems are unduly complex because of the wide fluctuation in signal levels generated as a result of the reception of light at the photodetectors after it has passed through the sample sites; this prevents efficient utilization of the overall dynamic range of amplification for the signal amplifiers of the measurement system.
Another limitation of conventional microplate reading devices is their inability to make useful quantitative measurements for ELISA protocols performed in filter-bottom microplates. A principal problem is that the individual wells in such plates vary considerably in their initial optical density relative to air thereby introducing considerable error when end-point measurements are taken. Kinetic analysis, on the other hand, is not affected by this type of problem.
Another major problem associated with conventional microplate reading devices, when used for assaying chromogenic reactions kinetically, is that they are subject to errors arising from erratic redistribution of the colored product as a result of phase separation and/or uncontrolled bulk movement of the aqueous phase of the sample during kinetic analysis. More specifically, in the case of ELISA protocols, where the enzyme is bound to the plastic surface of the microplate wells (on the bottom and/or part way up the sides), the bound enzyme interacts with an unstirred aqueous phase layer which typically causes localized phase separation of the colored product of the enzyme reaction due to its high local concentration. This separation introduces an unquantifiable error and a degree of non-linearity into such kinetic measurements. Even in cases where the colored product remains in true solution, erratic bulk movement of the aqueous phase leads to uneven redistribution of the concentrated product and hence to an unquantifiable error.